Assessment of low-intensity fluorescence signals in living cardiac cells using time-resolved laser spectroscopy

In the aim to study calcium distribution and dynamics in single living left-ventricular rat cardiomyocytes, we have applied a new method for discrimination of the calcium probe fluorescence signal from the intrinsically fluorescing cell constituents. In a case study investigating calcium-sensitive dye Fluo-3, we have characterized the spectral and the lifetime fingerprints of the Fluo-3 fluorescence and of the cell flavin autofluorescence by spectrally-resolved confocal microscopy and/or by multi-wavelength time-correlated single photon counting. We demonstrate that employing such spectral database, a low-intensity Fluo-3 signals comparable to the cell autofluorescence can be successfully resolved in cardiomyocytes using spectral linear unmixing algorithm.